Figure 4. Projection neurons connect the ANS to the synaptic nervous system.

(A) Connectivity map of all possible known neuropeptide-GPCR signaling, colors represent modules defined by randomized community detection analysis in Gephi and node size represents authority. (B) Reconstructed IN^RGWaand IN^NOS projection neurons (ANS) (shades of brown and cyan) in a full body transmission electron microscopy (ssTEM) dataset of a 3-day-old larva, shown against a framework of ANS neurons (light grey). Axons and dendrites appear as lines and cell body positions are represented by spheres. (C) ssTEM reconstruction of IN^RGWa projection neurons and Ser-h1 serotonergic neurons. Red spheres indicate IN^RGWa presynaptic sites. (D) ssTEM reconstruction of IN^RGWa projection neurons and a presynaptic ciliomotor neuron, cMN^vl. (E) ssTEM reconstruction of IN^RGWa neurons and a presynaptic sensory neuron, pygPD^bicil. (F) Synaptic connectivity graph of IN^RGWa, Ser-h1, pygPD^bicil, and cMN^vl neurons. Synaptic inputs from the synaptic nervous system to the IN^RGWa neuron are in red. Peptidergic inputs to the IN^RGWa neurons are indicated with dashed arrows. (G) ssTEM reconstruction of IN^RGWa projection neurons, Ser-h1 neurons, and the cholinergic ciliomotor MC neuron, anterior view (H) Confocal microscopy image of correlated pixels of GCaMP6s signal in a 2-day-old Platynereis larva after the addition of 25 µM D-achatin neuropeptide, anterior view. Cells showing correlated activity with the serotonergic neurons (red) and the MC cell (cyan) are shown. IN^RGWa-dcl2 could not be identified in this larva and is likely obscured by the MC cell, Ser-h1^l and/or IN^RGWa-dcl1. (I) Neuronal activity patterns of individually identified neurons in a 2-day-old larva treated with 25 µM achatin.

Figure 4—figure supplement 1. Spatial distribution of dense core vesicles and synapses in an RGWa-expressing projection interneuron, IN^RGW-dcr1.

(A) Reconstruction of 50 layers of TEM sections from the 72 hpf larva ssTEM dataset containing the axon of IN^RGWa-dcr1 in the larval neurosecretory plexus. The axon is coloured light blue, synaptic sites are red, and dense core vesicles purple. Scale bar = 2 µm. This reconstruction can be viewed in 3D in Video 3. (B–E) TEM sections from different z-positions in the 72 hpf larva ssTEM dataset containing the neurites of the IN^RGWa-dcr1 axon (blue). Examples of dense core vesicles are indicated by purple arrowheads, red arrows indicate synapses (clusters of small clear vesicles at the neurite border). Scale bar A: 2 µm; scale bar B-E: 500 nm.

Figure 4—figure supplement 2. (A–D) Standard deviation in fluorescent calcium signal of the (A) Ser-h1^l, (B) Ser-h1^r, (C) IN^RGWa-dcl1 and (D) IN^RGWa-dcr1 neurons in 2-day-old larvae before and after treatment with 25 µM D-achatin.

(E–H) Correlation of neuronal calcium signals of (E) Ser-h1^l, (F) Ser-h1^r, (G) IN^RGWa-dcl1 and (H) IN^RGWa-dcr1 with calcium signals measured from the MC cell in 2-day-old larvae before and after treatment with 25 µM D-achatin.